U.S. patent application number 14/610044 was filed with the patent office on 2015-05-28 for zinc chelating agents for depleting xiap and sensitizing tumor cells to apoptosis.
The applicant listed for this patent is Institute for Cancer Research d/b/a The Research Institute of Fox Chase Cancer Center, Institute for Cancer Research d/b/a The Research Institute of Fox Chase Cancer Center. Invention is credited to Seth M. Cohen, Vladimir Kolenko, Alexander Kutikov, Ervin Teper, Robert G. Uzzo.
Application Number | 20150147291 14/610044 |
Document ID | / |
Family ID | 50028454 |
Filed Date | 2015-05-28 |
United States Patent
Application |
20150147291 |
Kind Code |
A1 |
Kutikov; Alexander ; et
al. |
May 28, 2015 |
Zinc Chelating Agents for Depleting Xiap and Sensitizing Tumor
Cells to Apoptosis
Abstract
The invention provides zinc chelating agents and formulations
thereof that deplete XIAP in cancer cells and sensitize the cells
to apoptosis-inducing agents. The invention provides methods for
sensitizing apoptosis-resistant cancer cells to apoptosis-inducing
agents, as well as methods for treating a subject with a combined
therapy of a zinc chelating agent and an apoptosis-inducing
agent.
Inventors: |
Kutikov; Alexander;
(Voorhees, NJ) ; Kolenko; Vladimir; (Philadelphia,
PA) ; Cohen; Seth M.; (San Marcos, CA) ; Uzzo;
Robert G.; (Ambler, PA) ; Teper; Ervin;
(Huntington, PA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Institute for Cancer Research d/b/a The Research Institute of Fox
Chase Cancer Center |
Philadelphia |
PA |
US |
|
|
Family ID: |
50028454 |
Appl. No.: |
14/610044 |
Filed: |
January 30, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
PCT/US2013/052495 |
Jul 29, 2013 |
|
|
|
14610044 |
|
|
|
|
61677202 |
Jul 30, 2012 |
|
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Current U.S.
Class: |
424/85.1 ;
435/375 |
Current CPC
Class: |
A61K 31/4709 20130101;
A61K 31/4709 20130101; A61K 31/47 20130101; A61K 38/191 20130101;
A61K 45/06 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
424/85.1 ;
435/375 |
International
Class: |
A61K 31/47 20060101
A61K031/47; A61K 38/19 20060101 A61K038/19 |
Goverment Interests
STATEMENT OF GOVERNMENT SUPPORT
[0002] The inventions described herein were made, in part, with
funds obtained from the National Institutes of Health, Grant Nos.
R01-CA108890 and R01-CA134463, and from the U.S. Department of
Defense, Grant No. W81XWH-10-1-018. The U.S. government may have
certain rights in these inventions.
Claims
1. A method for inducing apoptosis in an apoptosis-resistant cell,
comprising contacting an apoptosis-resistant cell with a compound
selected from the group consisting of Formula I, ##STR00009##
Formula II, ##STR00010## Formula V, ##STR00011## wherein X1 is H,
Cl, or F and X2 is CN or F, and a pharmaceutically acceptable salt
thereof, in an amount of effective to deplete X-linked inhibitor of
apoptosis protein (XIAP) in the cell, and contacting the cell with
an amount of an apoptosis-inducing agent effective to induce
apoptosis in the cell.
2. The method of claim 1, wherein the compound is Formula I,
##STR00012## or a pharmaceutically acceptable salt thereof.
3. The method of claim 1, wherein the compound is Formula II,
##STR00013## or a pharmaceutically acceptable salt thereof.
4. The method of claim 1, wherein the compound is Formula V,
##STR00014## wherein X1 is H, Cl, or F and X2 is CN or F, or a
pharmaceutically acceptable salt thereof.
5. The method of claim 4, wherein X1 is Cl and X2 is F.
6. The method of claim 1, wherein the apoptosis-inducing agent is
TNF-related apoptosis-inducing ligand (TRAIL).
7. The method of claim 1, wherein the cell is a tumor cell.
8. The method of claim 7, wherein the tumor cell is a prostate
tumor cell.
9. The method of claim 8, wherein the prostate tumor cell is a
castration resistant prostate tumor cell.
10. The method of claim 1, wherein the compound substantially
depletes XIAP in the cell.
11. A method for treating a tumor, comprising administering to a
subject in need thereof a compound selected from the group
consisting of Formula I, ##STR00015## Formula II, ##STR00016##
Formula V, ##STR00017## wherein X1 is H, Cl, or F and X2 is CN or
F, and a pharmaceutically acceptable salt thereof, in an amount of
effective to deplete X-linked inhibitor of apoptosis protein (XIAP)
XIAP in cells of the tumor, and administering to the subject an
apoptosis-inducing agent in an amount of effective to induce
apoptosis in cells of the tumor in which XIAP has been depleted,
thereby treating the tumor.
12. The method of claim 11, wherein the compound is Formula I,
##STR00018## or a pharmaceutically acceptable salt thereof.
13. The method of claim 11, wherein the compound is Formula II,
##STR00019## or a pharmaceutically acceptable salt thereof.
14. The method of claim 11, wherein the compound is Formula V,
##STR00020## wherein X1 is H, Cl, or F and X2 is CN or F, or a
pharmaceutically acceptable salt thereof.
15. The method of claim 14, wherein X1 is Cl and X2 is F.
16. The method of claim 11, wherein the apoptosis-inducing agent is
TNF-related apoptosis-inducing ligand (TRAIL).
17. The method of claim 11, wherein the tumor is an
apoptosis-resistant tumor.
18. The method of claim 11, wherein the tumor is a tumor of the
prostate gland.
19. The method of claim 18, wherein the tumor of the prostate gland
is a castration resistant tumor of the prostate gland.
20. The method of claim 11, wherein the compound substantially
depletes XIAP in cells of the tumor.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of International
Application No. PCT/US2013/052495, filed on Jul. 29, 2013, and
claims priority to U.S. Provisional Application No. 61/677,202,
filed on Jul. 30, 2012, the contents of each application are
incorporated by reference herein, in their entirety and for all
purposes.
FIELD OF THE INVENTION
[0003] The invention relates generally to the field of cancer
treatment. More particularly, the invention relates to combination
therapies for treating cancer cells, and especially for enhancing
the susceptibility of cancer cells to cytotoxic agents by depleting
proteins that support resistance to cell death.
BACKGROUND OF THE INVENTION
[0004] Various publications, including patents, published
applications, technical articles and scholarly articles are cited
throughout the specification. Each of these cited publications is
incorporated by reference herein, in its entirety and for all
purposes.
[0005] Castration-resistant prostate cancer (CRPC) is the second
leading cause of cancer death among men in the United States.
Current treatment options, including docetaxel-based chemotherapy
and more recently Sipuleucel-T, have been shown to improve survival
by only 2 to 4 months. Furthermore, androgen ablation--a mainstay
treatment for advanced prostate cancer--is coming under increasing
scrutiny due to the well-documented reduction in the quality of
life, unclear impact on survival, and increasingly better
understood ill-effects on cardiovascular health. Development of new
therapeutic strategies for advanced prostate cancer represents an
urgent need in today's clinical landscape.
SUMMARY OF THE INVENTION
[0006] The invention provides compounds of Formula I
##STR00001##
or a pharmaceutically acceptable salt thereof. As well, the
compound may be comprised in a composition with a pharmaceutically
acceptable carrier.
[0007] The invention also provides compounds of Formula II
##STR00002##
or a pharmaceutically acceptable salt thereof. As well, the
compound may be comprised in a composition with a pharmaceutically
acceptable carrier.
[0008] The invention also provides compounds of Formula V
##STR00003##
or a pharmaceutically acceptable salt thereof, wherein X1 is H, Cl,
or F; and X2 is CN, or F. In some preferred aspects of formula V,
X1 is Cl and X2 is F. In some preferred aspect of Formula V, X1 is
H and X2 is CN. As well, the compound may be comprised in a
composition with a pharmaceutically acceptable carrier.
[0009] The compounds of Formula I, Formula II, and/or Formula V,
the respective pharmaceutically acceptable salts thereof, and
compositions comprising these compounds or pharmaceutically
acceptable salts may be used in various methods. In some aspects, a
method comprises depleting X-linked inhibitor of apoptosis protein
(XIAP) in a cell, and comprises contacting the cell with an amount
of a compound having Formula I or a pharmaceutically acceptable
salt thereof, effective to deplete XIAP in the cell. In some
aspects, a method comprises depleting XIAP in a cell, and comprises
contacting the cell with an amount of a compound having Formula II
or a pharmaceutically acceptable salt thereof, effective to deplete
XIAP in the cell. In some aspects, a method comprises depleting
XIAP in a cell, and comprises contacting the cell with an amount of
a compound having Formula V or a pharmaceutically acceptable salt
thereof, effective to deplete XIAP in the cell. The compound of
Formula I or Formula II or Formula V may be comprised in a
composition with a pharmaceutically acceptable carrier. The cell is
preferably a tumor cell, including a cell of a tumor of the
prostate gland. The tumor of the prostate gland may be castration
resistant. The XIAP is preferably depleted by about 90% to about
99%, relative to the level of a cell of the same type that has not
been contacted with Formula I, Formula II, a respective
pharmaceutical salt thereof, or a composition thereof. XIAP
depletion preferably comprises degradation of XIAP protein.
[0010] In some aspects, a method comprises chelating zinc in a
cell, and comprises contacting the cell with an amount of a
compound having Formula I, or a pharmaceutically acceptable salt
thereof, effective to chelate zinc in the cell. In some aspects, a
method comprises chelating zinc in a cell, and comprises contacting
the cell with an amount of a compound having Formula II, or a
pharmaceutically acceptable salt thereof, effective to chelate zinc
in the cell. In some aspects, a method comprises chelating zinc in
a cell, and comprises contacting the cell with an amount of a
compound having Formula V, or a pharmaceutically acceptable salt
thereof, effective to chelate zinc in the cell. The compound of
Formula I or Formula II or Formula V may be comprised in a
composition with a pharmaceutically acceptable carrier. The cell is
preferably a tumor cell, including a cell of a tumor of the
prostate gland. The tumor of the prostate gland may be castration
resistant.
[0011] In some aspects, a method comprises enhancing the
sensitivity of a tumor cell to an apoptosis-inducing agent, and
comprises depleting XIAP in the tumor cell to a level at which the
cell is sensitive to the apoptosis-inducing agent, and the step of
depleting XIAP comprises contacting the cell with an amount of a
compound having Formula I, or a pharmaceutically acceptable salt
thereof, effective to deplete XIAP in the cell to the level at
which the cell is sensitive to the apoptosis-inducing agent. In
some aspects, a method comprises enhancing the sensitivity of a
tumor cell to an apoptosis-inducing agent, and comprises depleting
XIAP in the tumor cell to a level at which the cell is sensitive to
the apoptosis-inducing agent, and the step of depleting XIAP
comprises contacting the cell with an amount of a compound having
Formula II, or a pharmaceutically acceptable salt thereof,
effective to deplete XIAP in the cell to the level at which the
cell is sensitive to the apoptosis-inducing agent. In some aspects,
a method comprises enhancing the sensitivity of a tumor cell to an
apoptosis-inducing agent, and comprises depleting XIAP in the tumor
cell to a level at which the cell is sensitive to the
apoptosis-inducing agent, and the step of depleting XIAP comprises
contacting the cell with an amount of a compound having Formula V,
or a pharmaceutically acceptable salt thereof, effective to deplete
XIAP in the cell to the level at which the cell is sensitive to the
apoptosis-inducing agent. The compound of Formula I or Formula II
or Formula V may be comprised in a composition with a
pharmaceutically acceptable carrier. The tumor cell is preferably a
cell of a tumor of the prostate gland. The tumor of the prostate
gland may be castration resistant. The tumor cell may be resistant
to the apoptosis-inducing agent in the absence of XIAP depletion.
The apoptosis-inducing agent may comprise TRAIL, or an
apoptosis-inducing derivative thereof. XIAP depletion preferably
comprises degradation of XIAP protein.
[0012] In some aspects, a method comprises inducing apoptosis in an
apoptosis-resistant cell, and comprises contacting the cell with an
amount of a compound having Formula I, or a pharmaceutically
acceptable salt thereof, effective to deplete XIAP in the cell, and
contacting the cell with an amount of an apoptosis-inducing agent
effective to induce apoptosis in the cell. In some aspects, a
method comprises inducing apoptosis in an apoptosis-resistant cell,
and comprises contacting the cell with an amount of a compound
having Formula II, or a pharmaceutically acceptable salt thereof,
effective to deplete XIAP in the cell, and contacting the cell with
an amount of an apoptosis-inducing agent effective to induce
apoptosis in the cell. In some aspects, a method comprises inducing
apoptosis in an apoptosis-resistant cell, and comprises contacting
the cell with an amount of a compound having Formula V, or a
pharmaceutically acceptable salt thereof, effective to deplete XIAP
in the cell, and contacting the cell with an amount of an
apoptosis-inducing agent effective to induce apoptosis in the cell.
The apoptosis-resistant cell is preferably resistant to the
apoptosis-inducing agent, and the agent preferably comprises TRAIL
or an apoptosis-inducing derivative thereof. The compound of
Formula I or Formula II or Formula V may be comprised in a
composition with a pharmaceutically acceptable carrier. The
apoptosis-resistant cell is preferably a tumor cell of a tumor of
the prostate gland. The tumor of the prostate gland may be
castration resistant. The tumor cell may be resistant to the
apoptosis-inducing agent in the absence of XIAP depletion. The XIAP
is preferably depleted by about 90% to about 99%, relative to the
level of a cell of the same type that has not been contacted with
Formula I, Formula II, or Formula V, a respective pharmaceutical
salt thereof, or a composition thereof. XIAP depletion preferably
comprises degradation of XIAP protein.
[0013] The invention also features methods for treating an
apoptosis-resistant tumor in a subject in need thereof. In some
aspects, the methods comprise administering to the subject an
amount of a compound having Formula I, or a pharmaceutically
acceptable salt thereof, effective to deplete XIAP in cells of the
tumor, and administering to the subject an amount of an
apoptosis-inducing agent effective to induce apoptosis in cells of
the tumor in which XIAP has been depleted. In some aspects, the
methods comprise administering to the subject an amount of a
compound having Formula II, or a pharmaceutically acceptable salt
thereof, effective to deplete XIAP in cells of the tumor, and
administering to the subject an amount of an apoptosis-inducing
agent effective to induce apoptosis in cells of the tumor in which
XIAP has been depleted. In some aspects, the methods comprise
administering to the subject an amount of a compound having Formula
V, or a pharmaceutically acceptable salt thereof, effective to
deplete XIAP in cells of the tumor, and administering to the
subject an amount of an apoptosis-inducing agent effective to
induce apoptosis in cells of the tumor in which XIAP has been
depleted. The compound of Formula I or Formula II or Formula V may
be comprised in a composition with a pharmaceutically acceptable
carrier. The tumor is preferably a tumor of the prostate gland,
which may be castration resistant. The XIAP is preferably depleted
by about 90% to about 99% in cells of the tumor. The
apoptosis-inducing agent may comprise TRAIL, or an
apoptosis-inducing derivative thereof. The subject is preferably a
human being. XIAP depletion preferably comprises degradation of
XIAP protein.
[0014] The invention also features kits. The kits may be used to
practice any of the methods described or exemplified herein. In
some aspects, the kits comprise a compound having Formula I, or a
pharmaceutically acceptable salt thereof, in an amount effective to
deplete XIAP in a cell, an amount of TRAIL or an apoptosis-inducing
derivative thereof effective to induce apoptosis in a XIAP-depleted
cell, and instructions for using the compound or pharmaceutically
acceptable salt thereof and the TRAIL or apoptosis-inducing
derivative thereof in the method, for example, a method for
enhancing the sensitivity of a tumor cell to an apoptosis-inducing
agent, for example, TRAIL, or a method for inducing apoptosis in an
apoptosis-resistant cell, or a method for treating an
apoptosis-resistant tumor in a subject in need thereof. In some
aspects, the kits comprise a compound having Formula II, or a
pharmaceutically acceptable salt thereof, in an amount effective to
deplete XIAP in a cell, an amount of TRAIL or an apoptosis-inducing
derivative thereof effective to induce apoptosis in a XIAP-depleted
cell, and instructions for using the compound or pharmaceutically
acceptable salt thereof and the TRAIL or apoptosis-inducing
derivative thereof in the method, for example, a method for
enhancing the sensitivity of a tumor cell to an apoptosis-inducing
agent, for example, TRAIL, or a method for inducing apoptosis in an
apoptosis-resistant cell, or a method for treating an
apoptosis-resistant tumor in a subject in need thereof. In some
aspects, the kits comprise a compound having Formula V, or a
pharmaceutically acceptable salt thereof, in an amount effective to
deplete XIAP in a cell, an amount of TRAIL or an apoptosis-inducing
derivative thereof effective to induce apoptosis in a XIAP-depleted
cell, and instructions for using the compound or pharmaceutically
acceptable salt thereof and the TRAIL or apoptosis-inducing
derivative thereof in the method, for example, a method for
enhancing the sensitivity of a tumor cell to an apoptosis-inducing
agent, for example, TRAIL, or a method for inducing apoptosis in an
apoptosis-resistant cell, or a method for treating an
apoptosis-resistant tumor in a subject in need thereof. The kit may
comprise a pharmaceutically acceptable carrier into which the
compound of Formula I or Formula II or Formula V, and/or the TRAIL
or an apoptosis-inducing derivative thereof may be mixed.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1 shows treatment with protoporphyrin IX (PPIX) or
5-Ala reduces XIAP levels in prostate cancer cells. PC-3 cells were
cultured in medium alone, or in the presence of PPIX (50 .mu.M) for
4 hours, or 5-Ala (250 .mu.M) for 24 hours. Expression of XIAP and
GAPDH (control) was detected by immunoblotting with an antibody to
XIAP or GADPH, respectively.
[0016] FIG. 2 shows that treatment with 5-Ala augments HGS-ETR1 the
antitumor effect in an ectopic mouse model of prostate cancer. PC-3
tumors were established in male C.B17/Icr-scid mice. When tumor
volumes reach approximately 200 mm.sup.3, the animals were grouped
(n=5 for each group) and treated with saline (control), an ip
injection of 5-Ala (10 mg/mouse, daily) and/or an iv injection of
HGS-ETR1 (0.25 mg/mouse once a week).
[0017] FIG. 3 shows a Western blot measuring the effect of various
zinc chelating agents on the expression of XIAP; Parallel cell
cultures were treated with TPEN as a positive control for zinc
chelation. Beta-actin was screened as a negative control.
[0018] FIG. 4 shows a comparison of select chelating agents of
against TPEN in the depletion of cellular levels of XIAP in
castration-resistant prostate cancer cells.
DETAILED DESCRIPTION OF THE INVENTION
[0019] Various terms relating to aspects of the present invention
are used throughout the specification and claims. Such terms are to
be given their ordinary meaning in the art, unless otherwise
indicated. Other specifically defined terms are to be construed in
a manner consistent with the definition provided herein.
[0020] As used herein, the singular forms "a," "an," and "the"
include plural referents unless expressly stated otherwise.
[0021] The term "about" as used herein encompasses variations of
plus or minus 25%, 20%, 15%, 10%, 5%, 1%, 0.5%, 0.25%, or 0.1% from
the specified value.
[0022] The terms subject and patient are used interchangeably
herein. A subject may be any animal, including a mammal such as a
farm animal (e.g., horse, cow, sheep, pig), laboratory animal
(e.g., mouse, rat, rabbit), companion animal (e.g., dog, cat), or
non-human primates (e.g., new world monkey and old world monkey).
In highly preferred aspects, the subject is a human being.
[0023] MR13 comprises a compound having Formula I, and all
pharmaceutically acceptable salts thereof. Formula I:
##STR00004##
[0024] MR44 comprises a compound having Formula II, and all
pharmaceutically acceptable salts thereof. Formula II:
##STR00005##
[0025] MR66 comprises a compound having Formula II, and all
pharmaceutically acceptable salts thereof. Formula III:
##STR00006##
[0026] MR96 comprises a compound having Formula II, and all
pharmaceutically acceptable salts thereof. Formula IV:
##STR00007##
[0027] Formula V comprises a compound having Formula V, and all
pharmaceutically acceptable salts thereof. Formula V:
##STR00008##
wherein X1 is H, Cl, or F; and X2 is CN, or F. In some preferred
aspects of formula V, X1 is Cl and X2 is F. In some preferred
aspect of Formula V, X1 is H and X2 is CN.
[0028] Pharmaceutically acceptable salts of Formula I, Formula II,
and Formula V may be acid or base salts. Non-limiting examples of
pharmaceutically acceptable salts include sulfates, methosulfates,
methanesulfates, pyrosulfates, bisulfates, sulfites, bisulfites,
nitrates, besylates, phosphates, monohydrogenphosphates,
dihydrogenphosphates, metaphosphates, pyrophosphates, chlorides,
bromides, iodides, fluorides, acetates, propionates, decanoates,
caprylates, acrylates, formates, isobutyrates, caproates,
heptanoates, propiolates, oxalates, malonates, succinates,
suberates, sebacates, fumarates, maleates, dioates, benzoates,
chlorobenzoates, methylbenzoates, dinitromenzoates,
hydroxybenzoates, methoxybenzoates, phthalates, sulfonates,
toluenesulfonates, xylenesulfonates, pheylacetates,
phenylpropionates, phenylbutyrates, citrates, lactates,
.gamma.-hydroxybutyrates, glycollates, tartrates,
methanesulfonates, propanesulfonates, mandelates, and other salts
customarily used or otherwise U.S. FDA-approved.
[0029] Depleting includes reducing, decreasing, blocking,
preventing, delaying, stopping, and/or downregulating the biologic
activity or expression of a molecule or pathway of interest. By way
of example, but not of limitation, depleting XIAP protein includes
reducing detectable levels of XIAP, which may include degrading
XIAP protein in a cell.
[0030] Anticancer therapeutic regimens trigger tumor cell death
largely through the induction of apoptosis. Recent experiments
demonstrated that treatment of prostate cancer cells with the zinc
chelating agent N,N,N',N'-tetrakis(2-pyridylmethyl)-ethylenediamine
(TPEN) induces selective and rapid depletion of the X-linked
inhibitor of apoptosis protein (XIAP), a gatekeeper molecule that
determines apoptosis resistance in tumor cells of various origins.
Other studies have demonstrated that down-regulation of XIAP
results in marked sensitization of cancer cells to drug-mediated
apoptosis (Cheung H H et al. (2006) Clin. Cancer Res. 12:3238-42;
Schimmer A D, et al. (2006) Cell Death Differ. 13:179-88; and
Schimmer A D et al. (2004) Cancer Cell 5:25-35). It is believed
that coupling delivery of zinc chelating compounds with that of a
chemo- and/or immunotherapeutic agents may markedly increase the
therapeutic index of a given agent.
[0031] The process whereby a normal prostate epithelial cell
transforms into a cancerous cell involves the loss of the cell's
ability to accumulate intracellular zinc. This loss of zinc
accumulation is one of the most consistent and persistent
characteristics of prostatic malignancy. Zinc-deficient cancer
cells are more sensitive to zinc depletion compared with normal
zinc-accumulating cells.
[0032] It has been observed in accordance with the invention that
novel zinc chelating agents of Formula I and Formula II induce
selective and rapid depletion of XIAP in prostate cancer cells at
least as effectively as TPEN. In some cases, XIAP was completely or
nearly completely depleted. In addition, it has been observed that
these chelating agents, when combined with TNF-related
apoptosis-inducing ligand (TRAIL), induced significant levels of
apoptosis in TRAIL-resistant prostate cancer cells. The level of
apoptosis with a combination of the chelating agent and TRAIL was
6-8 fold higher than the level of apoptosis observed from the
chelator alone, and upwards of 16-fold higher than the level of
apoptosis observed from TRAIL alone. Accordingly, the invention
features zinc chelating agents of Formula I, Formula II, and
Formula V, compositions comprising these agents, kits comprising
these agents, and methods of using these agents for inducing cell
death in cancer cells. The methods may be carried out in vivo, in
vitro, or in situ.
[0033] A composition may comprise a zinc chelator of Formula I, or
a pharmaceutically acceptable salt thereof, and a carrier. A
composition may comprise a zinc chelator of Formula II, or a
pharmaceutically acceptable salt thereof, and a carrier. A
composition may comprise a zinc chelator of Formula V, or a
pharmaceutically acceptable salt thereof, and a carrier. A carrier
may comprise a pharmaceutically acceptable carrier.
Pharmaceutically acceptable carriers include any that do not
interfere (or substantially interfere) with the biological activity
of the Formula I, Formula II, or Formula V compound, including zinc
chelating activity, and preferably is not toxic to the subject to
which it is administered. Pharmaceutically acceptable carriers
include aqueous vehicles such as water, alcohol (e.g., ethanol or
glycol), saline solutions, dextrose solutions, and balanced salt
solutions, or a physiologically compatible buffer, such as Hanks's
solution, Ringer's solution, or physiological saline buffer, as
well as nonaqueous vehicles such as alcohols and oils, including
plant or vegetable-derived oils such as olive oil, cottonseed oil,
corn oil, canola oil, sesame oil, and other non-toxic oils. The
carrier may contain formulatory agents, such as suspending,
stabilizing and/or dispersing agents. The compositions may comprise
one or more pharmaceutically acceptable excipients.
[0034] The compositions preferably comprise a therapeutically
effective amount of the compound such as a compound having Formula
I, Formula II, Formula V, or pharmaceutically acceptable salt of
Formula I, Formula II, or Formula V. A therapeutically effective
amount may comprise an amount effective to chelate zinc in a tumor
cell, an amount effective to enhance sensitivity of a tumor cell to
an apoptosis-inducing agent, an amount effective to induce
apoptosis in an apoptosis-resistant tumor cell, or an amount
effective to treat an apoptosis-resistant tumor cell in a subject
in need thereof. The compositions may be prepared to provide from
about 0.05 mg to about 1000 mg of the compound (Formula I, Formula
II, or Formula V), or pharmaceutically acceptable salt thereof. The
compositions may comprise from about 1 mg to about 200 mg of the
compound, may comprise from about 10 mg to about 200 mg of the
compound, may comprise from about 10 mg to about 100 mg of the
compound, may comprise from about 50 mg to about 100 mg of the
compound, may comprise from about 50 mg to about 250 mg of the
compound, may comprise from about 50 mg to about 400 mg of the
compound, may comprise from about 100 mg to about 200 mg of the
compound, may comprise from about 100 mg to about 300 mg of the
compound, may comprise about 200 mg to about 250 mg of the
compound, may comprise about 225 mg to about 325 mg of the
compound, may comprise about 300 mg to about 400 mg of the
compound, may comprise about 400 mg to about 600 mg of the
compound, may comprise about 500 mg to about 750 mg of the
compound, may comprise about 500 mg to about 1000 mg of the
compound, may comprise about 600 mg to about 700 mg of the
compound, and, may comprise from about 750 mg to about 900 mg of
the compound, or pharmaceutically acceptable salt thereof. The
compositions may comprise from about 1 mg, about 50 mg, about 100
mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about
450 mg, about 500 mg, about 550 mg, about 600 mg, about 650 mg,
about 700 mg, about 750 mg, about 800 mg, about 850 mg, about 900
mg, about 950 mg, or about 1000 mg of the compound or
pharmaceutically acceptable salt thereof, though the composition
may comprise amounts lesser than about 1 mg or greater than about
1000 mg in some aspects. It is to be understood, however, that the
concentration may vary depending on the cell type, tumor type,
physical characteristics of the subject (species, age, height,
weight, gender, among others).
[0035] The compositions may be formulated for administration to a
subject in any suitable dosage form. The compositions may be
formulated for oral, buccal, nasal, transdermal, parenteral,
injectable, intravenous, subcutaneous, intramuscular, rectal, or
vaginal administrations. The compositions may be formulated in a
suitable controlled-release vehicle, with an adjuvant, or as a
depot formulation.
[0036] Preparations for parenteral administration include sterile
solutions ready for injection, sterile dry soluble products ready
to be combined with a solvent just prior to use, including
hypodermic tablets, sterile suspensions ready for injection,
sterile dry insoluble products ready to be combined with a vehicle
just prior to use and sterile emulsions.
[0037] Solid dosage forms include tablets, pills, powders, bulk
powders, capsules, granules, and combinations thereof. Solid dosage
forms may be prepared as compressed, chewable lozenges and tablets
which may be enteric-coated, sugar coated or film-coated. Solid
dosage forms may be hard or encased in soft gelatin, and granules
and powders may be provided in non-effervescent or effervescent
form. Solid dosage forms may be prepared for dissolution or
suspension in a liquid or semi-liquid vehicle prior to
administration.
[0038] Liquid dosage forms include aqueous solutions, emulsions,
suspensions, solutions and/or suspensions reconstituted from
non-effervescent granules and effervescent preparations
reconstituted from effervescent granules. Aqueous solutions
include, for example, elixirs and syrups. Emulsions may be oil-in
water or water-in-oil emulsions.
[0039] Pharmaceutically acceptable excipients utilized in solid
dosage forms include coatings, binders, lubricants, diluents,
disintegrating agents, coloring agents, flavoring agents,
preservatives, sweeteners, and wetting agents. Enteric-coated
tablets, due to their enteric-coating, resist the action of stomach
acid and dissolve or disintegrate in the neutral or alkaline
intestines. Other examples of coatings include sugar coatings and
polymer coatings. Sweetening agents are especially useful in the
formation of chewable tablets and lozenges. Pharmaceutically
acceptable excipients used in liquid dosage forms includes
solvents, suspending agents, dispersing agents, emulsifying agents,
surfactants, emollients, coloring agents, flavoring agents,
preservatives, and sweeteners.
[0040] Non-limiting examples of binders include glucose solution,
acacia mucilage, gelatin solution, sucrose and starch paste.
Non-limiting examples of lubricants include talc, starch, magnesium
or calcium stearate, lycopodium and stearic acid. Non-limiting
examples of diluents include lactose, sucrose, starch, kaolin,
salt, mannitol and dicalcium phosphate. Non-limiting examples of
disintegrating agents include corn starch, potato starch,
bentonite, methylcellulose, agar and carboxymethylcellulose.
Non-limiting examples of emulsifying agents include gelatin,
acacia, tragacanth, bentonite, and surfactants such as
polyoxyethylene sorbitan monooleate. Non-limiting examples of
suspending agents include sodium carboxymethylcellulose, pectin,
tragacanth, veegum and acacia.
[0041] Non-limiting examples of coloring agents include any of the
approved certified water soluble FD and C dyes, mixtures thereof,
and water insoluble FD and D dyes suspended on alumina hydrate.
Non-limiting examples of sweetening agents include dextrose,
sucrose, fructose, lactose, mannitol and artificial sweetening
agents such as saccharin, aspartame, sucralose, acelsulfame
potassium, and other artificial sweeteners. Non-limiting examples
of flavoring agents include synthetic flavors and natural flavors
extracted from plants such as fruits and mints, and synthetic
blends of compounds which produce a pleasant sensation.
Non-limiting examples of wetting agents include propylene glycol
monostearate, sorbitan monooleate, diethylene glycol monolaurate
and polyoxyethylene laural ether. Non-limiting examples of
enteric-coatings include fatty acids, fats, waxes, shellac,
ammoniated shellac and cellulose acetate phthalates. Non-limiting
examples of film coatings include hydroxyethylcellulose, sodium
carboxymethylcellulose, polyethylene glycol 4000 and cellulose
acetate phthalate. Non-limiting examples of preservatives include
glycerin, methyl and propylparaben, ethylparaben, butylparaben,
isobutylparaben, isopropylparaben, benzylparaben, citrate, benzoic
acid, sodium benzoate and alcohol.
[0042] Elixirs include clear, sweetened, hydroalcoholic
preparations. Pharmaceutically acceptable carriers used in elixirs
include solvents. Syrups include concentrated aqueous solutions of
a sugar, for example, sucrose, and may contain a preservative. An
emulsion is a two-phase system in which one liquid is dispersed
throughout another liquid. Pharmaceutically acceptable carriers
used in emulsions may include emulsifying agents and preservatives.
Suspensions may use pharmaceutically acceptable suspending agents
and preservatives. Pharmaceutically acceptable substances used in
non-effervescent granules, to be reconstituted into a liquid oral
dosage form, include diluents, sweeteners and wetting agents.
Pharmaceutically acceptable substance used in effervescent
granules, to be reconstituted into a liquid oral dosage form,
include organic acids and a source of carbon dioxide. Sources of
carbon dioxide include sodium bicarbonate and sodium carbonate.
Coloring and flavoring agents may be used in all such dosage
forms.
[0043] Additional excipients that may be included in any dosage
forms include, but are not limited to antimicrobial agents,
isotonic agents, buffers, antioxidants, local anesthetic agents,
sequestering or chelating agents, analgesic agents, antiemetic
agents, and other agents to enhance selected characteristics of the
formulation.
[0044] Antimicrobial agents may be cidal or static, and may be
antimicrobial, antifungal, antiparasitic, or antiviral.
Non-limiting examples of commonly used antimicrobial agents include
phenols or cresols, mercurials, benzyl alcohol, chlorobutanol,
methyl and propyl p-hydroxybenzoic acid esters, thimerosal,
benzalkonium chloride and benzethonium chloride. Acidic or basic pH
may be used for antimicrobial effects in some aspects. Non-limiting
examples of isotonic agents include sodium chloride and dextrose.
Non-limiting examples of buffers include phosphate and citrate
buffers.
[0045] The compositions may also be formulated in sustained release
vehicles or depot preparations. For example, the compositions may
be formulated with suitable polymeric or hydrophobic materials (for
example, as an emulsion in an acceptable oil) or ion exchange
resins, or as sparingly soluble derivatives, for example, as a
sparingly soluble salt. Liposomes, micelles, and emulsions are
well-known examples of such delivery vehicles.
[0046] The invention also features methods for depleting XIAP in a
cell. In some aspects, the methods comprise contacting the cell
with an amount of a compound having Formula I, or a
pharmaceutically acceptable salt thereof, effective to deplete XIAP
in the cell. In some aspects, the methods comprise contacting the
cell with an amount of a compound having Formula II, or a
pharmaceutically acceptable salt thereof, effective to deplete XIAP
in the cell. In some aspects, the methods comprise contacting the
cell with an amount of a compound having Formula V, or a
pharmaceutically acceptable salt thereof, effective to deplete XIAP
in the cell. Formula I or Formula II or Formula V may be comprised
in a composition.
[0047] The cell is preferably a tumor cell. The tumor cell may be a
tumor cell of the pancreas, a tumor cell of the head and neck, a
tumor cell of the lung, a tumor cell of the kidney, a tumor cell of
the breast, a tumor cell of the colon, a tumor cell of the ovary, a
tumor cell of a lymph node, a tumor cell of the stomach, a tumor
cell of the esophagus, a tumor cell of the skin, a tumor cell of
the brain, a tumor cell of the oral cavity, a tumor cell of the
pharynx, a tumor cell of the thyroid, a tumor of the adrenal gland,
a leukemia cell, a sarcoma cell, a tumor cell of the testes, a
tumor cell of the bladder, or a tumor cell of the prostate gland.
Prostate gland tumor cells are highly preferred, including but not
limited to castration-resistant prostate tumor cells. The cell may
be in a cell culture. The cell may be present in or otherwise
comprise a tumor tissue.
[0048] XIAP depletion may be measured according to the level of
XIAP depletion in a cell of the same type that has not been
contacted with Formula I, Formula II, or Formula V, or
pharmaceutically acceptable salt thereof. In some aspects, XIAP
depletion may be measured relative to the normal amount of XIAP in
the cell, and what is normal may vary according to a healthy
(non-cancerous) cell, or a tumor cell (including a tumor cell of a
stage I, stage II, stage III, or stage IV tumor), and/or the
particular cell type. Where a cell is a tumor cell, the normal
amount of XIAP in the cell is not necessarily the amount of XIAP in
a healthy, non-tumor cell. XIAP depletion may be measured according
to any technique suitable in the art, and may be measured at the
gene (e.g., mRNA) level, and/or the protein level. Without
intending to be limited to any particular theory or mechanism of
action, it is believed that zinc chelation induces degradation of
the XIAP protein, acting post-translationally.
[0049] The amount of XIAP depletion may comprise depletion of about
20% to about 100% of the level of XIAP in the cell. The amount of
XIAP depletion may comprise depletion of about 50% to about 99% of
the level of XIAP in the cell. The amount of XIAP depletion may
comprise depletion of about 50% to about 95% of the level of XIAP
in the cell. The amount of XIAP depletion may comprise depletion of
about 60% to about 80% of the level of XIAP in the cell. The amount
of XIAP depletion may comprise depletion of about 70% to about 90%
of the level of XIAP in the cell. The amount of XIAP depletion may
comprise depletion of about 75% to about 99% of the level of XIAP
in the cell. The amount of XIAP depletion may comprise depletion of
about 80% to about 95% of the level of XIAP in the cell. The amount
of XIAP depletion may comprise depletion of about 85% to about 99%
of the level of XIAP in the cell. The amount of XIAP depletion may
comprise depletion of about 85% to about 95% of the level of XIAP
in the cell. The amount of XIAP depletion may comprise depletion of
about 90% to about 99% of the level of XIAP in the cell. The amount
of XIAP depletion may comprise depletion of about 90% to about 95%
of the level of XIAP in the cell. The amount of XIAP depletion may
comprise depletion of about 90% to about 100% of the level of XIAP
in the cell. The amount of XIAP depletion may comprise depletion of
about 95% to about 99% of the level of XIAP in the cell. The amount
of XIAP depletion may comprise depletion of about 95% to about 100%
of the level of XIAP in the cell. The amount of XIAP depletion may
comprise about 50%, about 55%, about 60%, about 65%, about 70%,
about 75%, about 80%, about 81%, about 82%, about 83%, about 84%,
about 85%, about 86%, about 87%, about 88%, about 89%, about 90%,
about 91%, about 92%, about 93%, about 94%, about 95%, about 96%,
about 97%, about 98%, about 99%, or about 100% of the XIAP in the
cell. XIAP may be substantially depleted. Greater than about 50% of
XIAP may be depleted in the cell. Greater than about 60% of XIAP
may be depleted in the cell. Greater than about 70% of XIAP may be
depleted in the cell. Greater than about 75% of XIAP may be
depleted in the cell. Greater than about 80% of XIAP may be
depleted in the cell. Greater than about 85% of XIAP may be
depleted in the cell. Greater than about 90% of XIAP may be
depleted in the cell. Greater than about 91% of XIAP may be
depleted in the cell. Greater than about 92% of XIAP may be
depleted in the cell. Greater than about 93% of XIAP may be
depleted in the cell. Greater than about 94% of XIAP may be
depleted in the cell. Greater than about 95% of XIAP may be
depleted in the cell. Greater than about 96% of XIAP may be
depleted in the cell. Greater than about 97% of XIAP may be
depleted in the cell. Greater than about 98% of XIAP may be
depleted in the cell. Greater than about 99% of XIAP may be
depleted in the cell.
[0050] In some aspects, the invention provides methods for
enhancing the sensitivity of a tumor cell to an apoptosis-inducing
agent. The methods generally comprise depleting XIAP in the cell
and/or chelating zinc in the cell. Prostate gland tumor cells are
highly preferred, including but not limited to castration-resistant
prostate tumor cells. The cell may be in a cell culture. The cell
may comprise a tumor tissue. The cell may be resistant or
substantially resistant, as measured according to any suitable
technique or standard, to the apoptosis-inducing agent. Thus, for
example, the methods may sensitize a cell that is resistant to
apoptosis (as normally induced by contact with an effective amount
of the agent) caused by the agent, such that upon zinc chelation,
the cell will undergo apoptosis when contacted with the agent, or
will undergo apoptosis when contacted with lesser amounts of the
agent, or will undergo apoptosis when contacted with amounts of the
agent to which the cell is resistant.
[0051] XIAP is preferably depleted by chelating zinc in the cell.
Zinc is preferably chelated by contacting the cell with an amount
of a compound having Formula I, Formula II, or Formula V, or a
pharmaceutically acceptable salt thereof, effective to chelate zinc
in the cell. In some aspects, the methods comprise contacting the
cell with an amount of a compound having Formula I, or a
pharmaceutically acceptable salt thereof, effective to chelate zinc
in the cell. In some aspects, the methods comprise contacting the
cell with an amount of a compound having Formula II, or a
pharmaceutically acceptable salt thereof, effective to chelate zinc
in the cell. In some aspects, the methods comprise contacting the
cell with an amount of a compound having Formula V, or a
pharmaceutically acceptable salt thereof, effective to chelate zinc
in the cell. Formula I or Formula II or Formula V may be comprised
in a composition. The zinc is preferably zinc in ionic form. A
preferred zinc ion is the zinc (II) ion.
[0052] The amount of zinc chelated is preferably an amount
effective for sensitizing the cell to treatment with an
apoptosis-inducing agent such that the cell will undergo apoptosis,
or programmed cell death if contacted with an amount of the agent
effective to induce apoptosis. The amount of zinc chelated may be
an amount effective to deplete XIAP levels in the cell. The amount
of XIAP may be an amount described or exemplified herein. It is
believed that for those zinc chelators that possess a binding
constant that affords 1:1 zinc chelation, equimolar concentration
of chelator is required (e.g., for substantially all XIAP to be
depleted, substantially all cellular zinc needs to be bound), and
that for weaker zinc chelators, higher concentrations of zinc
chelators may be necessary.
[0053] The methods are suitable for sensitizing the cell to any
apoptosis-inducing agent to which the cell is resistant. In
preferred aspects, the methods sensitize the cells to TRAIL, or an
apoptosis-inducing analog, derivative, homolog, or variant of
TRAIL, or any agent that agonizes the TRAIL receptor to induce
apoptosis. Zinc chelation may sensitize a tumor cell to apoptosis
in response to any agent that activates a cell death receptor,
non-limiting examples of which include Fas/CD95, TNF-alpha, TRAIL
(including Mapatumumab (HGS-ETR1), a human monoclonal antibody that
induces cancer-cell death in a highly targeted way by activating
the protein known as TRAIL receptor 1). XIAP depletion also
sensitizes cancer cells to various chemotherapeutic agents, and
thus may sensitize the cell to a chemotherapeutic agent (Schimmer A
D et al. (2006) Cell Death Differ. 13:179-188).
[0054] The invention also features methods for inducing apoptosis
in an apoptosis-resistant cell. In some aspects, the methods
comprise chelating zinc in the cell and contacting the cell with an
amount of an apoptosis-inducing agent effective to induce apoptosis
in the cell. In some alternative aspects, the methods comprise
depleting XIAP in the cell and contacting the cell with an amount
of an apoptosis-inducing agent effective to induce apoptosis.
[0055] In some aspects of the methods, chelating zinc may comprise
contacting the cell with an amount of a compound having Formula I,
or a pharmaceutically acceptable salt thereof, effective to chelate
zinc in the cell. In some aspects of the methods, chelating zinc
may comprise contacting the cell with an amount of a compound
having Formula II, or a pharmaceutically acceptable salt thereof,
effective to chelate zinc in the cell. In some aspects of the
methods, chelating zinc may comprise contacting the cell with an
amount of a compound having Formula V, or a pharmaceutically
acceptable salt thereof, effective to chelate zinc in the cell.
Formula I or Formula II or Formula V may be comprised in a
composition. The zinc is preferably zinc in ionic form. A preferred
zinc ion is the zinc (II) ion. The amount of zinc chelation
preferably is an amount sufficient to sensitize the cell to the
induction of apoptosis upon contact with the apoptosis-inducing
agent.
[0056] In some aspects of the methods, depleting XIAP may comprise
contacting the cell with an amount of a compound having Formula I,
or a pharmaceutically acceptable salt thereof, effective to deplete
XIAP in the cell. In some aspects of the methods, depleting XIAP
may comprise contacting the cell with an amount of a compound
having Formula II, or a pharmaceutically acceptable salt thereof,
effective to deplete XIAP in the cell. In some aspects of the
methods, depleting XIAP may comprise contacting the cell with an
amount of a compound having Formula V, or a pharmaceutically
acceptable salt thereof, effective to deplete XIAP in the cell.
Formula I or Formula II or Formula V may be comprised in a
composition comprising a pharmaceutically acceptable carrier. The
amount of XIAP depletion preferably is an amount sufficient to
sensitize the cell to the induction of apoptosis upon contact with
the apoptosis-inducing agent. The amount of XIAP depletion may be
an amount described or exemplified herein.
[0057] The cell is preferably a tumor cell. The tumor cell may be a
tumor cell of the pancreas, a tumor cell of the head and neck, a
tumor cell of the lung, a tumor cell of the kidney, a tumor cell of
the breast, a tumor cell of the colon, a tumor cell of the ovary, a
tumor cell of a lymph node, a tumor cell of the stomach, a tumor
cell of the esophagus, a tumor cell of the skin, a tumor cell of
the brain, a tumor cell of the oral cavity, a tumor cell of the
pharynx, a tumor cell of the thyroid, a tumor of the adrenal gland,
a leukemia cell, a sarcoma cell, a tumor cell of the testes, a
tumor cell of the bladder, or a tumor cell of the prostate gland.
Prostate gland tumor cells are highly preferred, including but not
limited to castration-resistant prostate tumor cells. The cell may
be in a cell culture. The cell may comprise a tumor tissue.
[0058] The apoptosis inducing agent may be any agent whose
apoptosis-inducing activity or capability is caused, facilitated,
or enhanced by chelating zinc and/or depleting XIAP in the cell.
The agent is preferably TRAIL, or an apoptosis-inducing analog,
homolog, derivative, or variant of TRAIL, or any TRAIL receptor
agonist that induces apoptosis in the cell. Zinc chelation may
sensitize a tumor cell to apoptosis in response to any agent that
activates a cell death receptor, non-limiting examples of which
include Fas/CD95, TNF-alpha, TRAIL (including Mapatumumab
(HGS-ETR1), a human monoclonal antibody that induces cancer-cell
death in a highly targeted way by activating the protein known as
TRAIL receptor 1). The agent may be in a composition comprising a
pharmaceutically acceptable carrier
[0059] In accordance with the methods, the cell may be resistant or
substantially resistant to the apoptosis-inducing agent in the
absence of zinc chelation and/or XIAP depletion. Thus, for example,
upon zinc chelation and/or XIAP depletion the cell will undergo
apoptosis when contacted with the agent, or will undergo apoptosis
when contacted with lesser amounts of the agent, or will undergo
apoptosis when contacted with amounts of the agent to which the
cell is resistant (e.g., resistant when intracellular zinc is not
chelated or when XIAP levels are not depleted).
[0060] The methods are a combination therapy, in which the cell is
contacted with an agent to chelate zinc and/or to deplete XIAP, and
contacted with an agent to induce apoptosis. The zinc chelating
agent or XIAP-depleting agent may be contacted with the cell before
the cell is contacted with the apoptosis-inducing agent, or
substantially at the same time as the cell is contacted with the
apoptosis-inducing agent, although in some aspects, the agent may
be contacted with the cell after the cell is contacted with the
apoptosis-inducing agent.
[0061] In one detailed aspect, the method comprises contacting the
cell with an amount of a compound having Formula I, or
pharmaceutically acceptable salt thereof, effective to chelate zinc
in the cell, and contacting the cell with an amount of TRAIL
effective to induce apoptosis in the cell. In one detailed aspect,
the method comprises contacting the cell with an amount of a
compound having Formula II, or pharmaceutically acceptable salt
thereof, effective to chelate zinc in the cell, and contacting the
cell with an amount of TRAIL effective to induce apoptosis in the
cell. In one detailed aspect, the method comprises contacting the
cell with an amount of a compound having Formula V, or
pharmaceutically acceptable salt thereof, effective to chelate zinc
in the cell, and contacting the cell with an amount of TRAIL
effective to induce apoptosis in the cell. In one detailed aspect,
the method comprises contacting the cell with an amount of a
compound having Formula I, or pharmaceutically acceptable salt
thereof, effective to deplete XIAP in the cell, and contacting the
cell with an amount of TRAIL effective to induce apoptosis in the
cell. In one detailed aspect, the method comprises contacting the
cell with an amount of a compound having Formula II, or
pharmaceutically acceptable salt thereof, effective to deplete XIAP
in the cell, and contacting the cell with an amount of TRAIL
effective to induce apoptosis in the cell. In one detailed aspect,
the method comprises contacting the cell with an amount of a
compound having Formula V, or pharmaceutically acceptable salt
thereof, effective to deplete XIAP in the cell, and contacting the
cell with an amount of TRAIL effective to induce apoptosis in the
cell. The cell is preferably a prostate cancer cell. The prostate
cancer cell may be a castration-resistant prostate cancer cell. The
prostate cancer cell may be resistant to TRAIL.
[0062] The invention also features methods for treating
apoptosis-resistant tumor in a subject in need thereof. In some
aspects, the methods comprise administering to the subject an
amount of a zinc chelating agent effective to chelate zinc in cells
of the tumor, and administering to the subject an amount of an
apoptosis-inducing agent effective to induce apoptosis in cells of
the tumor in which zinc has been chelated by the zinc chelating
agent. In some aspects, the methods comprise administering to the
subject an amount of a XIAP depleting agent effective to deplete
XIAP in cells of the tumor, and administering to the subject an
amount of an apoptosis-inducing agent effective to induce apoptosis
in cells of the tumor in which XIAP has been depleted by the XIAP
depleting agent. Inducing apoptosis causes cells of the tumor to
die, thereby treating the tumor.
[0063] The methods may comprise administering to the subject an
amount of a compound having Formula I, or a pharmaceutically
acceptable salt thereof, effective to chelate zinc in cells of the
tumor (zinc chelating agent). The methods may comprise
administering to the subject an amount of a compound having Formula
I, or a pharmaceutically acceptable salt thereof, effective to
deplete XIAP in cells of the tumor (XIAP depleting agent). The
methods may comprise administering to the subject an amount of a
compound having Formula II, or a pharmaceutically acceptable salt
thereof, effective to chelate zinc in cells of the tumor (zinc
chelating agent). The methods may comprise administering to the
subject an amount of a compound having Formula II, or a
pharmaceutically acceptable salt thereof, effective to deplete XIAP
in cells of the tumor (XIAP depleting agent). The methods may
comprise administering to the subject an amount of a compound
having Formula V, or a pharmaceutically acceptable salt thereof,
effective to chelate zinc in cells of the tumor (zinc chelating
agent). The methods may comprise administering to the subject an
amount of a compound having Formula V, or a pharmaceutically
acceptable salt thereof, effective to deplete XIAP in cells of the
tumor (XIAP depleting agent). Formula I or Formula II or Formula V
may be comprised in a composition comprising a pharmaceutically
acceptable carrier.
[0064] The methods may comprise administering to the subject an
amount of TRAIL effective to induce apoptosis in cells of the tumor
in which zinc has been chelated and/or in which XIAP has been
deleted. The agent may comprise an apoptosis-inducing analog,
homolog, derivative, or variant of TRAIL. The TRAIL may be in a
composition comprising a pharmaceutically acceptable carrier. Zinc
chelation may sensitize a tumor cell to apoptosis in response to
any agent that activates a cell death receptor, non-limiting
examples of which include Fas/CD95, TNF-alpha, TRAIL (including
Mapatumumab (HGS-ETR1), a human monoclonal antibody that induces
cancer-cell death in a highly targeted way by activating the
protein known as TRAIL receptor 1), and such an agent may be in a
composition comprising a pharmaceutically acceptable carrier.
[0065] In one detailed aspect, the method comprises administering
to the subject an amount of a compound having Formula I, or
pharmaceutically acceptable salt thereof, effective to chelate zinc
in cells of the tumor, and administering to the subject an amount
of TRAIL effective to induce apoptosis in cells of the tumor in
which zinc has been chelated. In one detailed aspect, the method
comprises administering to the subject an amount of a compound
having Formula II, or pharmaceutically acceptable salt thereof,
effective to chelate zinc in cells of the tumor, and administering
to the subject an amount of TRAIL effective to induce apoptosis in
cells of the tumor in which zinc has been chelated. In one detailed
aspect, the method comprises administering to the subject an amount
of a compound having Formula V, or pharmaceutically acceptable salt
thereof, effective to chelate zinc in cells of the tumor, and
administering to the subject an amount of TRAIL effective to induce
apoptosis in cells of the tumor in which zinc has been chelated. In
one detailed aspect, the method comprises administering to the
subject an amount of a compound having Formula I, or
pharmaceutically acceptable salt thereof, effective to deplete XIAP
in cells of the tumor, and administering to the subject an amount
of TRAIL effective to induce apoptosis in cells of the tumor in
which XIAP has been deleted. In one detailed aspect, the method
comprises administering to the subject an amount of a compound
having Formula II, or pharmaceutically acceptable salt thereof,
effective to deplete XIAP in cells of the tumor, and administering
to the subject an amount of TRAIL effective to induce apoptosis in
cells of the tumor in which XIAP has been deleted. In one detailed
aspect, the method comprises administering to the subject an amount
of a compound having Formula V, or pharmaceutically acceptable salt
thereof, effective to deplete XIAP in cells of the tumor, and
administering to the subject an amount of TRAIL effective to induce
apoptosis in cells of the tumor in which XIAP has been deleted. The
cell is preferably a prostate cancer cell. The prostate cancer cell
may be a castration-resistant prostate cancer cell. The prostate
cancer cell may be resistant to TRAIL.
[0066] Each agent (zinc chelating, XIAP depleting, and/or apoptosis
inducing) may be administered to the subject according to any
technique suitable in the art. Each agent may be administered
directly to or at least in the proximity of the tumor of interest.
Administration may be by way of the blood or other biological
fluid. Each agent may be specifically targeted to the tumor in the
subject, for example, by using carriers, liposomes, antibodies,
and/or magnets. Any suitable route or technique to administer the
agent may be used, and may vary, for example, according to the
needs of the investigator or according to the characteristics of
the agent itself.
[0067] The apoptosis-resistant tumor preferably is resistant to the
apoptosis-inducing agent, such that chelating zinc and/or depleting
XIAP sensitizes the tumor to apoptosis from the apoptosis-inducing
agent. The tumor may be any tumor in which zinc accumulation
(especially the loss of zinc accumulation), zinc depletion, and/or
the level of XIAP is/are a factor in apoptosis resistance. A tumor
of the prostate gland, including a castration-resistant tumor, is a
preferred target.
[0068] The invention also features kits. The kits may be used, for
example, to carry out any of the methods described or exemplified
herein. In some aspects, a kit comprises a zinc chelating agent
having Formula I, or a pharmaceutically acceptable salt thereof,
and may further comprise an apoptosis inducing agent, preferably
TRAIL, and instructions for using the zinc chelating agent and, if
present, the apoptosis inducing agent, in any of the methods
described or exemplified herein. For example, the instructions may
be for using the zinc chelating agent and, if present, the
apoptosis inducing agent in a method for chelating zinc in a tumor
cell. The instructions may be for using the zinc chelating agent
and, if present, the apoptosis inducing agent in a method for
depleting XIAP in a tumor cell. The instructions may be for using
the zinc chelating agent and, if present, the apoptosis inducing
agent in a method sensitizing a tumor cell to an apoptosis-inducing
agent, including a tumor cell that is resistant to the agent. The
instructions may be for using the zinc chelating agent and, if
present, the apoptosis inducing agent in a method for treating a
tumor of the prostate gland in a subject in need thereof.
[0069] In some aspects, a kit comprises a zinc chelating agent
having Formula II, or a pharmaceutically acceptable salt thereof,
and may further comprise an apoptosis inducing agent, preferably
TRAIL, and instructions for using the zinc chelating agent and, if
present, the apoptosis inducing agent, in any of the methods
described or exemplified herein. For example, the instructions may
be for using the zinc chelating agent and, if present, the
apoptosis inducing agent in a method for chelating zinc in a tumor
cell. The instructions may be for using the zinc chelating agent
and, if present, the apoptosis inducing agent in a method for
depleting XIAP in a tumor cell. The instructions may be for using
the zinc chelating agent and, if present, the apoptosis inducing
agent in a method sensitizing a tumor cell to an apoptosis-inducing
agent, including a tumor cell that is resistant to the agent. The
instructions may be for using the zinc chelating agent and, if
present, the apoptosis inducing agent in a method for treating a
tumor of the prostate gland in a subject in need thereof.
[0070] In some aspects, a kit comprises a zinc chelating agent
having Formula V, or a pharmaceutically acceptable salt thereof,
and may further comprise an apoptosis inducing agent, preferably
TRAIL, and instructions for using the zinc chelating agent and, if
present, the apoptosis inducing agent, in any of the methods
described or exemplified herein. For example, the instructions may
be for using the zinc chelating agent and, if present, the
apoptosis inducing agent in a method for chelating zinc in a tumor
cell. The instructions may be for using the zinc chelating agent
and, if present, the apoptosis inducing agent in a method for
depleting XIAP in a tumor cell. The instructions may be for using
the zinc chelating agent and, if present, the apoptosis inducing
agent in a method sensitizing a tumor cell to an apoptosis-inducing
agent, including a tumor cell that is resistant to the agent. The
instructions may be for using the zinc chelating agent and, if
present, the apoptosis inducing agent in a method for treating a
tumor of the prostate gland in a subject in need thereof.
[0071] The compounds described and exemplified herein (e.g.,
Formula I, Formula II, Formula V, and pharmaceutically acceptable
salts thereof), may be for use as a medicament. Formulations of the
compounds described and exemplified herein (e.g., Formula I,
Formula II, Formula V, and pharmaceutically acceptable salts
thereof together with a pharmaceutically acceptable carrier), may
be for use as a medicament. The compounds (e.g., Formula I, Formula
II, Formula V, and pharmaceutically acceptable salts thereof) and
formulations (e.g., Formula I, Formula II, Formula V, and
pharmaceutically acceptable salts thereof together with a
pharmaceutically acceptable carrier) may be for use in the
treatment of a tumor of the prostate gland. The compounds (e.g.,
Formula I, Formula II, Formula V, and pharmaceutically acceptable
salts thereof) and formulations (e.g., Formula I, Formula II,
Formula V, and pharmaceutically acceptable salts thereof together
with a pharmaceutically acceptable carrier) may be for use in the
treatment of an apoptosis-resistant tumor of the prostate gland.
The compounds (e.g., Formula I, Formula II, Formula V, and
pharmaceutically acceptable salts thereof) and formulations (e.g.,
Formula I, Formula II, Formula V, and pharmaceutically acceptable
salts thereof together with a pharmaceutically acceptable carrier)
may be for use in the treatment of a castration-resistant tumor of
the prostate gland.
[0072] The compounds (e.g., Formula I, Formula II, Formula V, and
pharmaceutically acceptable salts thereof) and formulations (e.g.,
Formula I, Formula II, Formula V, and pharmaceutically acceptable
salts thereof together with a pharmaceutically acceptable carrier),
in combination with an agent that induces apoptosis, such as TRAIL
or a TRAIL receptor agonist that induces apoptosis, may be for use
in the treatment of a tumor of the prostate gland. The compounds
(e.g., Formula I, Formula II, Formula V, and pharmaceutically
acceptable salts thereof) and formulations (e.g., Formula I,
Formula II, Formula V, and pharmaceutically acceptable salts
thereof together with a pharmaceutically acceptable carrier), in
combination with an agent that induces apoptosis, such as TRAIL or
a TRAIL receptor agonist that induces apoptosis, may be for use in
the treatment of an apoptosis-resistant tumor of the prostate
gland. The compounds (e.g., Formula I, Formula II, Formula V, and
pharmaceutically acceptable salts thereof), in combination with an
agent that induces apoptosis, such as TRAIL or a TRAIL receptor
agonist that induces apoptosis, and formulations (e.g., Formula I,
Formula II, Formula V, and pharmaceutically acceptable salts
thereof together with a pharmaceutically acceptable carrier) may be
for use in the treatment of a castration-resistant tumor of the
prostate gland.
[0073] The invention also features methods for screening compounds
for capability of depleting XIAP in a cell. The methods comprise
contacting a zinc deficient cell with a test compound, and
measuring the level of XIAP in the cells in the presence of the
test compound relative to the level of XIAP in the cells in the
absence of the test compound, wherein depletion of XIAP in the
presence of the test compound indicates that the test compound is
capable of depleting XIAP in the cell. The cell may be a prostate
cancer cell, including a castration-resistant prostate cancer cell.
The cell may be comprised in a cell culture.
[0074] The test compound is preferably a known or suspected zinc
chelating agent. A test compound may be any purified molecule,
substantially purified molecule, molecules that are one or more
components of a mixture of compounds, or a mixture of a compound
with any other material that can be analyzed using the methods
described herein (e.g., a composition). Test compounds can be
organic or inorganic chemicals, or biomolecules, and all fragments,
analogs, homologs, conjugates, and derivatives thereof.
Biomolecules include proteins, polypeptides, nucleic acids, lipids,
monosaccharides, polysaccharides, and all fragments, analogs,
homologs, conjugates, and derivatives thereof. Test compounds can
be of natural or synthetic origin, and can be isolated or purified
from their naturally occurring sources, or can be synthesized de
novo. Test compounds can be defined in terms of structure or
composition, or can be undefined. Test compounds can be an isolated
product of unknown structure, a mixture of several known products,
or an undefined composition comprising one or more compounds.
Non-limiting examples of undefined compositions include cell and
tissue extracts, growth medium in which cells have been cultured,
and fermentation broths. The test compound can be contacted with
the cell according to any means suitable in the art, and for any
suitable period of time. The test compound can be assessed at
multiple concentrations.
[0075] The screening method may further comprise contacting a
second cell with the test compound and an apoptosis-inducing agent,
for example, TRAIL, and determining the level of apoptosis in the
cell in the presence of the test compound relative to the level of
apoptosis in the cell in the absence of the test compound, wherein
any increase, preferably a statistically significant increase, in
apoptosis indicates that the test compound enhances the sensitivity
of the cell to apoptosis induced by the apoptosis-inducing agent.
The second cell may be resistant to the apoptosis-inducing agent.
The second cell may be a zinc-deficient cell. The second cell may
be a prostate cancer cell, including a castration-resistant
prostate cancer cell. The second cell may be comprised in a cell
culture.
[0076] The invention also features method for synthesizing a zinc
chelating pro-drug. The zinc chelating pro-drug will lack the
ability to chelate zinc until converted into active (drug) form.
Conversion into active form may be by treatment of the pro-drug
with a chemical or biomolecule that allows the drug to bind to
zinc. For example, it is preferred that the pro-drug comprises
moieties that are cleaved by esterases inside of a cell, whereupon
the esterase-mediated cleavage activates the pro-drug into drug
form.
[0077] The synthesizing method may comprise protecting zinc-binding
moieties on a zinc chelating agent with a benzyl protecting group
to form a benzyl-protected zinc chelating agent, and covalently
coupling the benzyl-protected zinc chelating agent to a second
benzyl-protected chelating with an amine-containing linking agent.
The covalent coupling may comprise covalently bonding a carboxyl
group on the zinc chelating agent to an amine group on the linking
agent, thereby forming a carbodiimide (EDC). The covalent coupling
may comprise forming a NHS ester bond between the zinc chelating
agent and the linking agent. Suitable linking agents are shown in
FIG. 6B.
[0078] The following examples are provided to describe the
invention in greater detail. They are intended to illustrate, not
to limit, the invention.
Example 1
Preliminary Results
[0079] It has been demonstrated that zinc-specific chelator
N,N,N',N'-tetrakis(2-pyridylmethyl)-ethylenediamine (TPEN) rapidly
depletes XIAP in human prostate cancer cells. Makhov P et al.
(2008) Cell Death Differ. 15:1745-51. TPEN exhibits specificity for
malignant but not for benign cells. It is believed that the
molecular mechanism for this specificity stems from blunted
regulation of zinc uptake transporters in malignant cells compared
with normal prostate epithelial cells. For instance, expression of
zinc uptake transporters is significantly increased in benign
tissues during low-zinc conditions, resulting in compensatory zinc
uptake. In contrast, the expression of zinc uptake transporters is
virtually undetectable in malignant prostate cells.
[0080] The inhibition of XIAP expression is selective, as TPEN has
no effect on the expression levels of other zinc-containing members
of IAP family, i.e., cIAP1, cIAP2 and surviving. As well, the XIAP
mRNA level is not reduced in TPEN-treated cells. Therefore, zinc
chelation by TPEN induces depletion of XIAP specifically at the
protein level. TPEN treatment also produces a rapid decrease of
XIAP protein levels in a xenograft animal model of human prostate
cancer. The action of immunotherapeutic agents such as TRAIL
Receptor-1 antibody (HGS-ETR1, mapatumumab) (Human Genome Sciences)
and docetaxel are markedly potentiated by TPEN. Importantly, the
sensitization to therapeutic agents is specific to malignant but
not to benign cells. This cytotoxic specificity for malignant cells
is consistent with experiments showing that XIAP is preferentially
degraded in malignant but not in benign cells upon zinc chelation
and stems from blunted regulation of zinc uptake transporters upon
carcinogenesis.
[0081] To examine whether zinc-binding agents structurally distinct
from TPEN are also capable of decreasing XIAP levels, cells were
treated with either protoporphyrin IX (PPIX), a naturally occurring
chelating molecule or its precursor 5-aminolevulinic acid (5-Ala).
As demonstrated in FIG. 1, both agents significantly reduced XIAP
expression in PC-3 cells. Importantly, combined 5-ALA and HGS-ETR1
treatment completely arrested tumor growth in xenograft animal
model (FIG. 2).
Example 2
Heterocyclic Zinc Chelators Materials and Methods
[0082] Synthesis of Heterocyclic Zn Chelators.
[0083] The entire library of zinc chelators was supplied by the
Department of Chemistry and Biochemistry from the University of
California, San Diego.
[0084] Cell Lines and Culture Conditions.
[0085] A PC-3 castration-resistant prostate cancer cell line was
obtained from the American Type and Culture Collection (Rockville,
Md.). PC-3 cells were maintained in complete cell culture medium
(RPMI 1640 medium (Bio-Whittaker, Walkersville, Md.) supplemented
with 10% FCS (Hyclone, Logan, Utah), gentamicin (50 mg/L), sodium
pyruvate (1 mM) and non-essential amino acids (0.1 mM)).
[0086] Analysis of XIAP Depletion in PC-3 Cell Line Following
Administration of Heterocyclic Zn Chelators.
[0087] PC-3 cells were cultured under three distinct conditions;
medium alone, with administration of TPEN (8 .mu.M) as a positive
control for 2 hours, and with administration of an individual Zn
chelators (MR) (50 .mu.M) for 4 hours.
[0088] Western blot analysis was performed to assess for level of
XIAP depletion. Whole cell lysates were prepared as described
previously (Kolenko V et al. (1999) J. Immunol. 163: 590-8).
Protein concentrations were measured with BCA protein assay
reagents (Pierce, Rockford, Ill.). Equivalent amounts of proteins
(20 .mu.g) were mixed with an equal volume of 2.times. Laemmli
sample buffer, boiled and resolved by electrophoresis in 10% sodium
dodecyl sulfate-polyacrylamide gels (SDS-PAGE). The proteins were
transferred from the gel to a nitrocellulose membrane using an
electroblotting apparatus (Bio-Rad) (15 V, 3 mA/cm2 for 24
minutes).
[0089] Membranes were then incubated in a blocking solution
containing 5% nonfat dry milk overnight to inhibit nonspecific
binding. The membranes were then incubated with specific antibody
(1-3 .mu.g/ml) for 2 hours. After washing in TRIS/0.1% Tween 20 for
30 minutes, membranes were incubated for another 30 minutes with
horseradish peroxidase-conjugated secondary antibody. The membranes
were then washed and developed with enhanced chemiluminescence (ECL
Western Blotting Kit, Amersham, Arlington Heights, Ill.).
[0090] Animal Studies.
[0091] Two 6 week-old male C.B17/Icr-scid mice were injected
intravenously with 100 .mu.l of MR13 chelator at final
concentration of 15 mM. Intravenous administration of MR13 was well
tolerated by both animals.
Example 3
XIAP Depletion by New Zinc Chelating Agents
[0092] In pursuit of identifying more effective and biologically
flexible zinc chelators, a versatile platform for the preparation
of high-affinity zinc chelators and prochelators has been
developed. The platform is based on bidentate ligands such as
hydroxypyrones, hydroxypyrothiones, hydroxypyridinones (HOPOs), and
hydroxypyridinethiones (HOPTOs), which are effective chelators of
the zinc(II) ion. By taking advantage of the well-established
coordination chemistry of these ligands with the zinc(II) ion, a
series of zinc(II) sequestering agents has been developed.
[0093] The ability of novel heterocyclic zinc chelating agents to
deplete cellular levels of XIAP in castration-resistant prostate
cancer cells was assessed. One hundred and twenty candidate zinc
chelating agents were tested in the screen. Some of these
heterocyclic zinc chelators induced complete depletion of XIAP
protein in PC-3 cells as demonstrated in FIG. 3. Actin was used to
demonstrate equal protein loading. TPEN was used as a positive
control. Notably, XIAP depletion was a reliable marker of ability
to promote TRAIL-mediated apoptosis in prostate cancer cells as
judged by the DNA fragmentation assay. As demonstrated in FIG. 4,
MR13 (Formula I) and MR44 (Formula II) agents completely depleted
XIAP and promoted TRAIL-mediated cell death in PC-3 cells.
Meanwhile, agents such as MR66 (Formula III) and MR96 (Formula IV),
failed to induce XIAP depletion and thus did not succeeded in
producing an apoptotic response.
[0094] The invention is not limited to the embodiments described
and exemplified above, but is capable of variation and modification
within the scope of the appended claims.
* * * * *